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MATERIALS AND METHODSCell Culture and Radioactive Labeling: Normal humanfibroblasts, HSBP were routinely grown in Dulbecco's modifiedmedium plus 10% fetal calf serum at 370 in an atmospherecontaining 5% CO2. Cells grown to a monolayer in 75 cm2flasks were subcultured at a ratio of 1:3. 3H-thymidine(s.a. 3 Ci mmol) at 0.03 pCi/ml or 32P ortho phosphate at0.5 pCi/ml were added and the cells grown to a monolayer.Cell Plating UV Irradiation and Density Labelling: Cellswere plated at 7.105 per 150 mm petri dish and forty hourslater UV irradiated. Some 3H labeled cells were sampled todetermine the dimers induced, others were incubated in mediumcontaining Fluorodeoxyuridine (0.25 ug/ml) and bromodeoxy-uridine (3 ug/ml). 32P prelabeled cells for repair replica-tion studies were similarly incubated except that 3H tlhjmidine(s.a. 55 Ci/mmol) at 5 uCi/ml was also included in the medium.Extraction of DNA, Separation of Replicated and Unrepli-cated Portions: Cells were lysed with 0.5 M EDTA, 0.5%sarkosyl plus 200 ug/ml Proteinase K (Merck), and left at 370for 8 hrs prior to phenol extraction. Cesium chloride (1.25g/ml) was dissolved in each sample and centrifuged in a 40rotor of a Beckman 5-50 at 30,000 rpm for 40 hours. Gradientswere collected as 30 fractions of 0.13 ml, 10 1 of eachfraction acid precipitated to determine the heavy/light andlight/light peaks, and these portions pooled and dialysedagainst .011 TrisHCl, .001M EDTA, .04M NaCl pH 8.0.Estimation of Pyrimidine Dimers: This was carried out bydetermining the number of UV endonuclease sensitive sites (7).Determination of Repair Replication: Unreplicated andreplicated DNA from 32P prelabeled cells was obtained asabove. The light strand from heavy/light DNA, and both lightstrands from light/light DNA were further purified by 2centrifugations in alkaline cesium chloride (8). -H activityfound banding with light 32P labeled strands was used as ameasure of repair.RESULTSThe Effect of UV on DNA Synthesis: Table 1 gives theamounts of DNA synthesised in cells during 12 or 24 hoursafter 5 or 10 J-m-2. Cells receiving 5 J-m-2 by 24 hourshave synthesized as much as unirradiated controls, but after10 J.m-2 only half this amount is made during the same period.